The present invention relates to a method and apparatus for monitoring a gaseous environment for the presence of cellular material; more particularly it relates to an apparatus that is capable of providing a measure of presence and/or numbers of cellular microorganisms, such as bacterial cells, in a large volume of air such as in a warehouse or production facility or in an open air location where bacterial presence is suspected. The method and apparatus of the invention are particularly provided for determining the likelihood of pathogenic or allergenic material being present in an environment by continuous on-line measurement of cell numbers. The latter format provides a continuous monitoring of an environment.
There is a military need for detection of the incidence of attack using biological materials, including attack using bacteria, e.g., in the form of cells or spores. Such need includes the capability to monitor the air some distance upwind of an asset site in order that sufficient warning may be given to personnel on that site that an attack with bacteria is imminent. In such circumstances it is required that monitoring be carried out continuously that is for a continuous period of time for any one monitoring device e.g. from several to several tens of hours.
There is a further need for determining the presence of pathogens in facilities such as hospitals and in manufacturing premises in which foodstuffs, sterile pharmaceuticals or physiological supplements are being placed in containers prior to use. Before a production run it is desirable that the sterility of the packing environment be checked for the presence of pathogens or less harmful bacteria that may be used as an indication ad likely presence of pathogens or allergens.
In both of these situations it is necessary to process a large volume of airs either because of the continuous nature of the measurement or because of the need to sample a significant amount of clean room or sterile warehouse air. Furthermore in both situations it is necessary to screen for a wide range of bacteria regardless of type, as the threat may-not be that of a known genus or species.
It is known to use the luminol reaction to analyse air for the presence of haematin, but this technology is susceptible to giving readings with inorganic materials and is limited to a detection limit of 10.sup.3 bacterial cells in theory as only 10.sup.-16 grams of haematin is extractable from the average bacterial cell. Metal sensitivity giving high backgrounds render this system unreliable in practice.
It is known to screen for the likely presence of bacteria by analysing samples for the presence of adenosine triphosphate. This is readily carried out using luciferase and luciferin agent whereupon the presence of ATP allows luciferase to catalyse the oxidation of luciferin with the resultant emission of light. Samples are loaded into a luminometer and the amount of light emitted used as a measure of the amount of bacteria present. In order to liberate as much ATP from any cells present it is known to add a detergent to the sample in order to lyse the-cells and release the ATP.
Although such biochemistry has been extensively utilised with individual samples derived by direct sampling of surfaces, liquids and solids, there has been little development of luminometry equipment suitable for monitoring bacteria in air.
JP 62093634 discloses a counter for microorganisms which draws in an air sample, collects the microorganisms from that and extracts ATP from them before assaying the ATP using a luminescent reaction. This device uses a 0.2.mu. membrane filter to collect microorganisms from the air in a batchwise fashion, with the membrane being periodically analysed by being passed to an extracted station. No details of the sensitivity of this equipment are given, but its performance is limited by the ability of the air pump to draw sufficient sample air across the membrane and by the time taken to process the membrane microorganism content.
JP 58122281 discloses a method for detecting bacteria in air again using luminescent reagents to assay ATP. This method extracts ATP using a Tris-EDTA liquid buffer heated to 100.degree. C. from samples of 10 of liters of air per minute-batched in 10 minute samples. Filters are required to eliminate dirt and dust and-these are described as essential to the method. A cooling tube is required in order to avoid increase of background noise-due to raising of temperature of photomultiplier tubes use to monitor luminescence. This apparatus also uses an `extractor` it draw air into it at 10s of liters per minute. The exact nature of this `extractor` is not clear.
It is known to use cyclone devices to capture particulates from air, these devices typically being electrically driven and producing particulate depleted and particulate concentrated fractions. It is known to use such devices for the purposes of obtaining aerosols and other particulates from air for later analysis. For example GB 2245024 describes a cyclone for collecting a large sample volume of biological materials from the air; SU 1546481 and SU 11911460 describe use of cyclones to provide a particulate sample which is used to seed nutrient holding vessels or plates for analysis while SU 916535 collects bacteria from such cyclone on a filter band and viruses in a lower section wherefrom they are used to infect experimental animals, it is also known to use virtual impactors to. collect airborne particulates, see e.g. U.S. Pat. No. 4,942,297 and U.S. Pat. No. 4,670,135.
Again, none of these systems are capable of continuous monitoring of air for the presence of bacteria, particularly small amounts of pathogenic bacteria. A particular problem is the variation in concentration of fluid output from the cyclone with changes in humidity of the air being sampled. With very high throughput the cylone can run almost dry and produce high readings from a relatively normal background input.
JP5184350 describes a system for counting of bacterial cells suspended in air which aims to shorten the determination time and improve the accuracy of the results. JP 60016598 describes an alternative device for detecting bacteria in an amount of air. JP 3112495 describes a filter system for the detection of different microorganisms floating in air. None of these devices are suitable for continuous operation.